The risk of defective glucose counterregulation is greatly increased following an episode of hypoglycemia, with a blunted sympathoadrenal response a primary indicator of hypoglycemia-associated autonomic failure (HAAF). How HAAF develops is unknown but may be linked to failure of central glucose sensing mechanisms. The overall objective of these in vivo studies is to delineate the mechanisms of glucose sensing, counterregulation, and brain function during hypoglycemia. The hypothesis of this application is that hypoglycemia-induced changes in VMH glucose metabolism contribute to HAAF and that these changes are induced by the increased noradrenergic activity in the VMH responsible for initiating counterregulatory responses. The research proposed herein will determine if (Aim 1) HAAF can be induced by changes in glucose metabolism occurring solely in the VMH, (Aim 2) hypoglycemia-induced changes in glycogen metabolism in the VMH contribute to the development of HAAF, and (Aim 3) HAAF is a consequence of hypoglycemia-induced nordrenergic activity in the VMH required for counterregulatory responses to hypoglycemia. A fourth specific aim is to describe the glucose metabolome in the VMH by using stable isotopes of glucose and GC/MS analysis. Changes in the metabolome during an initial and following recurrent episodes of hypoglycemia that lead to HAAF. Hypoglycemia-induced changes in glucose metabolism in the VMH will be compared to other brain areas to determine if they are unique to glucose sensing areas or represent a more global response by nervous tissue. Understanding hypoglycemia-induced changes in VMH glucose metabolism that negatively affect activation of counterregulatory responses to subsequent hypoglycemia is necessary to develop novel approaches to prevent hypoglycemia unawareness and HAAF. PUBLIC HEALTH RELEVANCE: A single episode of hypoglycemia will adversely affect the corrective responses to subsequent hypoglycemia and represents a significant barrier to effective management of diabetes. Understanding the hypoglycemia-induced changes in glucose metabolism that negatively impact counterregulatory responses to subsequent hypoglycemia in critical brain centers is necessary to develop novel approaches to prevent hypoglycemia- associated autonomic failure.